The three mammalian MAPRE proteins play important roles in the microtubule dynamics of the cell cytoskeleton, and facilitate the binding of multiple +TIP proteins to microtubule plus ends. Work conducted by the 1000 Genomes Project, the Exome Sequencing Project, and many other groups has led to the identification of non-conservative coding sequence allelic variants of the MAPRE proteins in the human population that result in the production of mutant MAPRE proteins. Disruption of MAPRE protein function could decrease cell signaling fidelity (how well a cell receives and transmits information) and microtubule function. The MAPRE proteins are related by descent from a common ancestor to the Bim1 protein in budding yeast. Here I designed and constructed mutant versions of Bim1 that contained mutations corresponding to known MAPRE variants and expressed them in yeast. The yeast pheromone response pathway was utilized as a model signaling system to test the effects of the MAPRE variants on cell signaling. The output of the pheromone response pathway, and therefore amount of signal received and transmitted by the cell, was quantified using fluorescent reporters under the control of a pheromone responsive promoter in yeast strains expressing the mutant versions of Bim1. By comparing the pheromone pathway output via fluorescence intensity of the mutant MAPRE strains to the output of the wildtype MAPRE strains, I was able to discern effects of the Bim1 mutants on cell signaling. Deletion of Bim1 resulted in increased variation in the pheromone pathway output. Also, a mutant MAPRE allele consisting of a C-terminal domain deletion resulted in diminished pathway output and increased output variation. Adverse effects on cell signaling could result in poor decision making and environmental response, potentially leading to the generation of disease. By better understanding the effects of allelic variants in the human population, we will be able to develop individualized health plans and therapies.

Anatomically modern humans and Neanderthals overlapped in time and space, and recent genomic studies show that limited amounts of mating between these two groups occurred. The goal of my project is to analyze genomic patterns of Neanderthal sequences that survive in present day individuals. Of particular interest are regions of the human genome where there are no surviving Neanderthal sequences, which suggest genetic incompatibilities or selective pressure between human and Neanderthal DNA. Specifically, I am focusing on a 16 Mb region on chromosome 7 in which no Neanderthal sequences persist in modern humans. Strikingly, this region includes the gene FOXP2, which is involved in speech and language. I am performing a number of computational analyses to catalog all sequence differences between modern humans and Neanderthals in this region, and prioritizing variants that are most likely to be functionally important using comparative and functional genomics data. Ultimately, the goal of my project is to narrow down the set of variants in this region that may contribute to uniquely human phenotypes.

Childhood Apraxia of Speech (CAS) is a rare, severe, persistent pediatric motor speech disorder of largely unknown genetic cause that affects the ability to acquire intelligible speech and coordinate the volitional production of sounds by sequencing muscle movements for the production of words. Developmental Dyslexia is a common and specific childhood learning disorder defined as a significant impairment in reading ability that cannot be explained by deficits in intelligence, learning opportunity, motivation, or sensory acuity. Various dyslexia studies suggest that slowed global processing speed, especially when sequential modes are involved, may be a brain-based endophenotype of dyslexia under genetic control. Impaired sequential processing, hence, may be a biomarker shared by CAS and Developmental Dyslexia. Our group recently identified a de novo heterozygous deletion of a single gene in a proband with severe CAS, raising the question whether similar deletions are causal in other cases as well. Here we report a genome-wide Single-Nucleotide Polymorphism (SNP) and CNV findings in a cohort of 20 affected individuals in nine multigenerational families with well-characterized CAS and six unrelated individuals with Developmental Dyslexia. All participants met clinical research criteria for CAS as part of a larger study on familial motor speech sound disorder or Developmental Dyslexia. The genome-wide copy-number variation study of the selected individuals was completed using the Illumina HumanOmniExpress-24 BeadChips and Human Exome BeadChips. Analysis of the SNP content and CNV for overlapping variations across the genomes of these affected individuals give us insight into genomic regions associated with the clinical phenotype of sequential processing deficit in multiple domains.

Chromosomal DNA synthesis begins at origins of replication, with some origins activating earlier than others during S-phase. Neither the mechanism by which origin activation time is determined nor the biological significance of this temporal program is fully understood. Interestingly centromeres, sites where kinetochore complexes form to correctly separate chromosomes during mitosis, replicate early. In the budding yeast Saccharomyces cerevisiae, centromeres actively promote their own early replication by advancing the activation time of their neighboring origins. We previously showed, in cells lacking the mitotic checkpoint, an artificially delayed, late replicating centromere causes a dramatic increase in chromosome instability. In this study, I am investigating the function of centromere-like regions (CLRs) in S. cerevisiae. These sequences can bind the centromeric histone (Cse4), especially when CSE4 is overexpressed. Under this condition, additional kinetochore proteins can build up and CLRs take on some of the properties of functional centromeres. Because the kinetochore protein Ctf19 recruits a replication initiation factor, I hypothesize that the centromere-mimicking DNA sequences are also capable of recruiting the kinetochore protein associated with early replication to advance the initiation time of nearby origins. To test this possibility, I will examine the effects of the CLR on replication origin activation using a plasmid that contains two identical origins. I will clone a CLR next to one of the origins and determine whether this origin is now the earlier activated origin on the plasmid when CSE4 is overexpressed. The CLR I will use resides in an early replicating part of the yeast genome. In a complementary experiment I will delete the genomic copy of this CLR and measure its effect on firing time of the adjacent origins. The results of this study will provide us deeper insights on how cells orchestrate when and where in their genome to start duplication.

Genetic conflict ensues between two parties that have an antagonistic relationship, where fitness gains in one party necessitate fitness losses in the other. As a result, there is constant selective pressure on both parties to maintain evolutionary dominance. One such conflict may exist internally between the mitochondrial and nuclear genome. This arises from the difference in DNA inheritance patterns between the two parties. Nuclear DNA is transmitted maternally and paternally in Mendelian fashion whereas mitochondrial DNA is only transmitted maternally, leaving males as an evolutionary dead end for mitochondrial DNA. This suggests that male-harming mutations could arise and fixate in mitochondrial DNA as long as those mutations are beneficial or neutral to female fitness. One way for males to counteract such mutations is for the nuclear DNA to evolve suppressors of these mutations. This conflict may not commonly exist in nature because males with mutant mitochondria would appear wild-type if their nuclear genomes contain suppressors of these mutations. To reveal if a conflict exists, we took an experimental evolution approach, using Drosophila melanogaster, to break the coevolution of mitochondrial DNA and Nuclear DNA and allow mitochondrial DNA to evolve on its own. After 34 generations, we performed a variety of assays and sequence analysis to identify any mitochondrial male harming mutations. We isolated a single mutation and an associated fertility decrease in males. We are currently trying to identify if any suppressors of this mutation exist in nuclear DNA of other strains of melanogaster by introducing the nuclear genome of these strains into a male with the mutation. We predict that if such nuclear genomes exist, then the fertility of mutant males should be restored when this nuclear genome is introduced. This study will provide an evolutionary insight into disease and defects of males associated with mitochondrial mutations.

Large chromosomal amplicons and deletions or copy number variation (CNV) are found in many different cancers. However, the inheritance, stability, and fixation of these amplicons and deletions within a population are widely debated and poorly understood. In an effort to better understand how the stability of this large chromosomal rearrangement, I analyzed the stability of a large segmental amplification in the yeast S. cerevisiae. This amplification is commonly observed in clones evolved under sulfate-limited conditions and contains the sulfate transporter SUL1. This amplification is an important adaptive strategy used by the cells to improve their ability to extract the limited supply of sulfate available in the media. High copy number of this gene confers a competitive fitness advantage over other clones that have only one copy of the gene. We used a GFP marker integrated next to SUL1 to differentiate green clones, which contain a single copy of the SUL1 gene, from “super green” clones that have multiple copies of the gene. A super green evolved clone was grown in steady state growth vessels for ~30 generations in media that is non-selective for adaptation and our selective media as a control. I monitored the population using flow cytometry and quantitative PCR to find clones that have lost the amplification. These green clones were isolated using flow sorting and then had their genome sequenced to look at the scar that was left behind. This tells us how efficiently the cells can remove amplifications without causing detrimental effects to their fitness.

Eukaryotic genomes contain many copies of ribosomal DNA (rDNA) encoding the RNA components of ribosomes, ranging from 150 tandem repeated copies in the budding yeast Saccharomyces cerevisiae to approximately 700 copies in diploid human cells.  Recent work in the Brewer/Raghuraman and Bedalov labs used S. cerevisiae to identify connections between rDNA copy number and cellular processes such as genome replication and replicative aging.  Based on their findings, I hypothesize that a cell with too many rDNA copies may have difficulty with genome-wide DNA replication, while a cell with too few copies will not be able to satisfy ribosome demand from protein synthesis.  What dictates the size of rDNA region remains unknown, but we hope to learn about the relationship between the size of rDNA region and DNA replication by identifying its genetic regulators.  A preliminary survey found single gene deletion strains with altered numbers of rDNA repeats, supporting the idea that rDNA copy number is under genetic control.  Encouraged by this result, I screened 400 mutants from S. cerevisiae deletion collection for rDNA copy number.  I also examined the putative link between rDNA size and longevity: half of the mutants chosen in this screen were reported to have longer replicative lifespan and the other half were randomly selected.  I found that the average rDNA copy number is not significantly different between the two groups, suggesting that the relationship between the size of rDNA region and replicative lifespan is not direct.  I did observe a notable enrichment for mutants with altered rDNA size in genes that have mitochondrial function, defects in cell cycle progression, and abnormal cytoskeleton phenotypes.  By continuous investigation and exploring the molecular mechanisms that govern rDNA copy number, I hope to gain a deeper understanding of how the rDNA region interacts with genome-wide DNA replication.